Medullary Thymic Epithelial Cell Antigen-presentation Assays

Medullary thymic epithelial cells (mTEC) are bona fide antigen-presenting cells that play a crucial role in the induction of T-cell tolerance. By their unique ability to express a broad range of tissue-restricted self-antigens, mTEC control the clonal deletion (also known as negative selection) of potentially hazardous autoreactive T cells and the generation of Foxp3+ regulatory T cells. Here, we describe a protocol to assess major histocompatibility complex (MHC) class II antigen-presentation capacity of mTEC to CD4+ T cells. We detail the different steps of thymus enzymatic digestion, immunostaining, cell sorting of mTEC and CD4+ T cells, peptide-loading of mTEC, and the co-culture between these two cell types. Finally, we describe the flow cytometry protocol and the subsequent analysis to assess the activation of CD4+ T cells. This rapid co-culture assay enables the evaluation of the ability of mTEC to present antigens to CD4+ T cells in an antigen-specific context. Key features • This protocol builds upon the method used by Lopes et al. (2018 and 2022) and Charaix et al. (2022). • This protocol requires transgenic mice, such as OTIIxRag2-/- mice and the cognate peptide OVA323–339, to assess mTEC antigen presentation to CD4+ T cells. • This requires specific equipment such as a Miltenyi Biotec AutoMACS® Pro Separator, a BD FACSAriaTM III cell sorter, and a BD® LSR II flow cytometer.

This protocol is used in: eLife (2022), DOI: 10.7554/eLife.69982Medullary thymic epithelial cells (mTEC) are bona fide antigen-presenting cells that play a crucial role in the induction of T-cell tolerance.By their unique ability to express a broad range of tissue-restricted self-antigens, mTEC control the clonal deletion (also known as negative selection) of potentially hazardous autoreactive T cells and the generation of Foxp3+ regulatory T cells.Here, we describe a protocol to assess major histocompatibility complex (MHC) class II antigen-presentation capacity of mTEC to CD4+ T cells.We detail the different steps of thymus enzymatic digestion, immunostaining, cell sorting of mTEC and CD4+ T cells, peptide-loading of mTEC, and the co-culture between these two cell types.Finally, we describe the flow cytometry protocol and the subsequent analysis to assess the activation of CD4+ T cells.This rapid co-culture assay enables the evaluation of the ability of mTEC to present antigens to CD4+ T cells in an antigen-specific context.

Background
The thymus is a primary lymphoid organ that ensures the production of functional and self-tolerant naïve T cells.The development of those cells depends on stromal niches composed of thymic epithelial cells (TEC) that provide essential cues for the survival, proliferation, differentiation, and migration of developing T cells, called thymocytes (Irla, 2022).According to their localization in the thymus, TEC are subdivided into two main subsets: cortical (cTEC) and medullary TEC (mTEC).cTEC support early stages of thymopoiesis, including T-cell progenitor homing, Tcell lineage commitment, proliferation and survival of immature thymocytes, and positive selection of CD4 + and CD8 + single positive thymocytes.In contrast, mTEC control late stages of T-cell development, i.e., the clonal deletion of highly autoreactive thymocytes and the generation of mature Foxp3 + regulatory T cells (Treg).mTEC are commonly subdivided into two main subsets based on the expression of MHC class II (MHC-II) and CD80 molecules: mTEC lo (MHC-II lo CD80 lo ) and mTEC hi (MHC-II hi CD80 hi ), with mTEC lo containing progenitors capable of differentiating into mTEC hi (Gray et al., 2006;Gäbler et al., 2007;Miragaia et al., 2018).mTEC constitute a crucial antigen reservoir due to their unique capacity to express a wide range of tissue-restricted self-antigens.The expression of the majority of these tissue-restricted self-antigens is mediated by the transcription factors Aire (Autoimmune regulator) and Fezf2 (Fez family zinc finger 2), which regulate the expression of 3,000-4,000 and 600-700 genes, respectively (Sansom et al., 2014;Takaba et al., 2015).mTEC are bona fide antigen-presenting cells, as they can efficiently induce the clonal deletion of highly autoreactive CD4 + and CD8 + T cells, as well as the differentiation of CD4 + T cells towards the Foxp3 + Treg cell lineage (Aschenbrenner et al., 2007;Hinterberger et al., 2010;Aichinger et al., 2013).Interestingly, our lab recently showed that Aire + mTEC also have the ability to restimulate recirculating Foxp3 + Treg upon their re-entry in the thymus in an antigen-specific manner (Charaix et al., 2022).It is important to notice that mTEC-CD4 + T cell interactions act as bidirectional signals, as not only mTEC are crucial for CD4 + T cell selection but also CD4 + T cells control mTEC differentiation (Irla et , 2022).This in vitro co-culture protocol describes the steps to analyze the antigen-presentation capacity of mTEC through the activation of CD4 + T cells in an antigen-specific manner.It describes the different steps of thymus collection, enzymatic digestion, immunostaining, cell sorting of mTEC and CD4 + T cells, peptide-loading of mTEC, and the co-culture between these two cell types.Although this approach may be limited by the fact that it is an in vitro assay, it has the advantage of specifically and rapidly evaluating, within two days, the antigen-presentation capacity of mTEC.This protocol can also be used to assess the antigen-presentation capacity of human mTEC or dendritic cell subsets, the latter also implicated in the induction of T-cell tolerance (Lopes et al., 2015;Irla, 2022).Verify that the mouse is unresponsive to a toe pinch.2. Place the mouse on its back and pin each limp down to the dissection board.Spray with 70% ethanol to sterilize the mouse body.3. Open the thoracic cavity with scissors and carefully expose the heart and thymus above without cutting major blood vessels.4. Gently remove the thymus with a pair of forceps.

Biological species
Note: Make sure to remove fat and connective tissue from the thymus.5. Transfer the thymus into a 15 mL collection tube pre-filled with 2 mL of enzymatic digestion buffer for WT mice or 2 mL of cold FACS buffer for OTIIxRag2 -/-mice.

B. Thymic epithelial cell isolation
1. Digest the thymus from WT mice in a water bath at 37 °C for 15 min in 2 mL of digestion buffer; then, dissociate the tissue by approximately 20 recurrent aspirations through a 1,000 μL tip.Note: Agitate the fractions gently during the digestion process and avoid bubbles.2. Filter thymic cells through a 70 μm pore cell strainer into a 50 mL tube with 3 mL of cold FACS buffer.3. Collect remnant undigested tissue from the cell strainer with forceps and return it to the 15 mL tube containing digestion buffer.4. Digest remnant tissue in a water bath at 37 °C for 15 min.Then, dissociate the tissue by approximately 20 recurrent aspirations through a 1,000 μL tip. 5. Filter thymic cells through a 70 μm pore cell strainer into the 50 mL tube with 3 mL of cold FACS buffer.6. Repeat steps B3-B5 until complete tissue digestion.7. Centrifuge at 450× g for 5 min at 4 °C .

D. In vitro co-culture assays
1.After the purification of mTEC, centrifuge the Eppendorf collection tubes at 450× g for 5 min at 4 °C .2. Remove the supernatant and carefully resuspend the cell pellet in culture medium supplemented with 5 μg/mL of OVA323-339 peptide.Note: OVA323-339 concentration can be adjusted as required.3. Distribute mTEC in a plate with U bottom (100 μL/well) according to the cell concentration needed for the experimental plan.4. Incubate the cells in a 37 °C , 5% CO2 air humidified incubator for 1 h. 5.After the purification of CD4 + T cells, centrifuge the Eppendorf collection tubes at 450× g for 5 min at 4 °C .

Data analysis
FACS data were acquired on a BD ® LSR II flow cytometer using violet laser 405 nm, blue laser 488 nm, green laser 561 nm, and red laser 633 nm.FACS data were then analyzed using FlowJo software (version 10.
8.1).The gating strategy used to identify the proportion of activated CD69 + OTII T cells after co-culture with OVA323-339-loaded mTEC is shown in Figure3A.This protocol can be used with different ratios of mTEC to OTII CD4 + T cells to assess the antigen-presentation capacity of mTEC.As depicted in Figure3B, the activation of OTII CD4 + T cells, reflected by the upregulation of CD69, gradually increases with the numbers of OVA323-339-loaded mTEC.The details regarding the analysis can be found in the original article, e.g., Figure 2A(Lopes et al., 2022).
Store up to one month at 4 °C and keep on ice for the duration of the protocol.Store up to one month at 4 °C and keep on ice for the duration of the protocol.Store up to one month at 4 °C .One hour before use, place the medium in a water bath at 37 °C.
Note: Enzymes are sensitive to temperature; therefore, prepare before use.